W 


IMAGE  EVALUATION 
TEST  TARGET  (MT-3) 


CIHM/ICMH 

Microfiche 

Series. 


CIHM/ICIVIH 
Collection  de 
microfiches. 


jt^- 


Canadian  Institute  for  Historical  IVIicroreproductions  /  Institut  Canadian  de  microreproductions  historiques 


m^ 


Technical  and  Bibliographic  Notes/Notes  techniques  et  bibliographiques 


The  Institute  has  attempted  to  obtain  the  best 
original  copy  available  for  filming.  Features  of  this 
copy  which  may  be  bibliographically  unique, 
which  may  alter  any  of  the  images  in  the 
reproduction,  or  which  may  significantly  change 
the  usual  method  of  filming,  are  checked  below. 


D 
D 
D 
D 
D 
D 
D 


Coloured  covers/ 
Couverture  de  couleur 

Covers  damaged/ 
Couverture  endommag6e 


Covers  restored  and/or  laminated/ 
Couverture  restaur6e  et/ou  pellicul6e 


D 


D 


Cover  title  missing/ 

Le  titre  de  couverture  manque 

Coloured  maps/ 

Cartes  g6ographiques  en  couleur 

Coloured  ink  (i.e.  other  than  blue  or  black)/ 
Encre  de  couleur  (i.e.  autre  que  bleue  ou  noire) 

Coloured  plates  and/or  illustrations/ 
Planches  et/ou  illustrations  en  couleur 

Bound  with  other  material/ 
Rali6  avec  d'autres  documents 

Tight  binding  may  cause  shadows  or  distortion 
along  interior  margin/ 

La  reliure  serrde  peut  causer  de  I'ombre  ou  de  la 
distortion  le  long  de  lb  marge  int6rieure 

Blank  leaves  added  during  restoration  may 
appear  within  the  text.  Whenever  possible,  these 
have  been  omitted  from  filming/ 
II  se  peut  que  certaines  pages  blanches  ajout6es 
lors  d'une  restauration  apparaissent  dans  le  texte, 
mais,  lorsque  cela  6tait  possible,  ces  pages  n'ont 
pas  6t6  film6es. 

Additional  comments:/ 
Commentaires  suppl6mentaires; 


L'Institut  a  microfilmd  le  meilleur  exemplaire 
qu'il  lui  a  6t6  possible  de  se  procurer.  Les  details 
de  cet  exemplaire  qui  sont  peut-Atre  uniques  du 
point  de  vue  bibliographique,  qui  peuvent  modifier 
une  image  reproduite,  ou  qui  peuvent  exiger  une 
modification  dans  ia  methods  normale  de  filmage 
sont  indiqu6s  ci-dessous. 

□   Coloured  pages/ 
Pages  de  couleur 

□    Pages  damaged/ 
Pages  endommag6es 

I — I    Pages  restored  and/or  laminated/ 


D 
D 


This  item  is  filmed  at  the  reduction  ratio  checked  below/ 

Ce  document  est  film6  au  taux  de  rMuction  indiqu6  ci-dessous. 


Pages  restaur^es  et/ou  pelliculdes 

Pages  discoloured,  stained  or  foxet 
Pages  ddcolordes,  tachet6es  ou  piqu6es 

Pages  detached/ 
Pages  ddtachdes 

Showthrough/ 
Transparence 

Quality  of  prir 

Quality  in6gale  de  I'impression 

Includes  supplementary  materia 
Comprend  du  materiel  suppldmentaire 


r — I  Pages  discoloured,  stained  or  foxed/ 

I      I  Pages  detached/ 

I      I  Showthrough/ 

I      I  Quality  of  print  varies/ 

|~n  Includes  supplementary  material/ 


Only  edition  available/ 
Seule  Edition  disponible 

Pages  wholly  or  partially  obscured  by  errata 
slips,  tissues,  etc.,  have  been  ref limed  to 
ensure  the  best  possible  image/ 
Les  pages  totalement  ou  partiellement 
obscurcies  par  un  feuillet  d'errata,  une  pelure, 
etc.,  ont  6t6  filmdes  d  nouveau  de  fapon  d 
obtenir  la  meilleure  image  possible. 


10X 

14X 

18X 

22X 

26X 

30X 

'i 
'i 

! 

i 

12X 

16X 

20X 

24X 

28X 

32X 

<  ' 


ills 

lu 

difier 

ine 

age 


The  copy  filmed  here  has  been  reproduced  thanks 
to  the  generosity  of: 

Library  of  Congress 
Photoduplication  Service 

The  images  appearing  here  are  the  best  quality 
possible  considering  the  condition  and  legibility 
of  the  original  copy  and  in  keeping  with  the 
filming  contract  specifications. 


Original  copies  In  printed  paper  covers  are  filmed 
beginning  with  the  front  cover  and  ending  on 
the  last  page  with  a  printed  or  illustrated  Impres- 
sion, or  the  back  cover  when  appropriate.  All 
other  original  copies  are  filmed  beginning  on  the 
first  page  with  a  printed  or  illustrated  impres- 
sion, and  ending  on  the  last  page  with  a  printed 
or  illustrated  impression. 


The  last  recorded  frame  on  each  microfiche 
shall  contain  the  symbol  ^^^  (meaning  "CON- 
TINUED"), or  the  symbol  V  (meaning  "END"), 
whichever  applies. 


L'exemplaire  film*  fut  reproduit  grAce  A  la 
gAnArosIt*  de: 

Library  of  Congress 
Photoduplication  Service 

Les  Images  sulvantes  ont  hxh  reproduites  avec  le 
plus  grand  soln,  compte  tenu  de  ia  condition  at 
de  la  nettetA  de  I'exemplalra  fiimi.  et  en 
conformity  avec  les  conditions  du  contrat  de 
fllmage. 

Les  exemplalres  orlglnaux  dont  la  couverture  en 
papier  est  ImprlmAe  sont  filmAs  en  commen^ant 
par  le  premier  plat  et  en  terminant  solt  par  la 
dernlAre  page  qui  comporte  une  empreinte 
d'Impresslon  ou  d'illustration,  solt  par  le  second 
plat,  salon  le  cas.  Tous  les  autres  exemplalres 
orlglnaux  sont  fllmAs  en  commenpant  par  ia 
premlAre  page  qui  comporte  une  empreinte 
d'impression  ou  d'illustration  et  en  terminant  par 
la  darnlAre  page  qui  comporte  une  telle 
empreinte. 

Un  des  symboles  sulvants  apparaltra  sur  ia 
dernlAre  Image  de  cheque  microfiche,  selon  le 
cas:  le  symbole  -^  signlfle  "A  SUIVRE",  le 
symbole  V  signlfle  "FIN". 


Maps,  plates,  charts,  etc.,  may  be  filmed  at 
different  reduction  ratios.  Those  too  large  to  be 
entirely  included  in  one  exposure  are  filmed 
beginning  in  the  upper  left  hand  corner,  left  to 
right  and  top  to  bottom,  as  many  frames  as 
required.  The  following  diagrams  illustrate  the 
method: 


Les  cartes,  planches,  tableaux,  etc.,  peuvent  dtre 
filmte  A  des  taux  de  reduction  diff^rents. 
Lorsque  le  document  est  trop  grand  pour  Atre 
reproduit  en  un  seul  cllchA,  II  est  flimA  A  partir 
de  Tangle  supArleur  gauche,  de  gauche  A  droite, 
et  de  haut  en  bas,  en  prenant  le  nombre 
d'Images  nAcessalre.  Les  diagrammes  sulvants 
illustrent  la  m^thode. 


rata 


elure. 


D 


32X 


1 

2 

3 

1 

2 

3 

4 

5 

6 

'>*« 


t  , 


,*  Cornell  TAnivcrsity . . 


DEPARTMENT  OF  BOTANY 


THE  DEVELOPMENT  OF  THE 

ARCHEGONIUM  AND  FERTILIZATION 

IN  THE  HEMLOCK  SPRUCE 

(Tsuga  Canadensis  Carr.) 


IBy  VXiUiam  Alpbonso  Aurrill 


A  Thesis  submitteci  to  the  University  Faculty  of 
Cornell  University  for  the  Degree  of  Doctor 
of  Philosophy^  June,  (900. 


(■    mAY  23  1Q0I 


^^... 


■^'soKiM  m^t 


ITHACA,  N.  Y. 


t^bmals  of£otar^ 


WAMurr.ll.del. 


MURRILL  —  TSUGA  CANADENSIS. 


^i. 


VoLXIV,  PIXXXI. 


IS. 


Universityr  Press,  Orford. 


-?:-^i'"as^ 


t 


if  i 


tn 


t/bmaZs  ofJBoteif^ 


■TO, 


WA.Murnll.del. 


MURRILL  —  TSUOA   CANADENSIS. 


y^^„-,,^gBafelt/«M^*^Wh^«  -^J- 


yoixiv,pi.xxm. 


40. 


UnivBrwty  Press,  Oxford. 


;i^itll!?VWMUi»«W'Niii. 


The  Development  of  the  Archegonium 

and  Fertilization  in  the  Hemlock  Spruce 

(Tsuga  canadensis,  Carr.). 


BY 


WILLIAM  A.  MURRILL*. 


With  Plate*  XXXI  and  XXXU. 


THE  material  for  these  studies  has  been  collected  with 
great  regularity  for  the  past  three  years  from  a  hemlock 
standing  alone  on  the  bank  of  a  stream  in  an  open  pasture 
near  the  University  grounds.  The  tree  is  well  advanced  in 
years  and  has  fruited  heavily  every  season.  The  female 
cones  are  terminal  on  the  larger  horizontal  twigs,  the  male 
cones  occurring  in  great  abundance  on  the  smaller  ones.  At 
pollination,  there  is  no  change  in  the  position  of  the  female 
cones ;  they  remain  slightly  pendent,  the  scales  opening 
a  little  and  receiving  the  pollen  from  below  as  it  floats 
upward.  Soon  after  pollination,  the  pedicels  lengthen  and 
the  cones,  which  are  now  considerably  heavier,  hang  directly 
downward.  Pollination  on  a  single  tree  occupies  about  three 
days,  but  a  week  or  more  elapses  before  it  is  completed  on 
all  the  trees  of  this  locality.  In  1899  it  was  at  its  maximum 
on  May  19,  in  1900  on  May  2  a.  The  seasons  frequently  differ 
more  than  this. 

'  Read  before  the  Botanical  Society  of  America,  at  its  sixth  Annual  Meeting  in 
New  York  City,  June  38,  1900. 

[Annala  of  Botany,  Vol.  ZIV.  No.  LVI.  December,  1900.] 


■I 


MWM^SBKvr- 


■msms»mmu»i.- — .•— 


i 


584  MnniU. —  The  Development  of  the  Archegonium 

Two  weeks  after  pollination  the  archcgonial  rudiments 
appear ;  a  week  later  the  necks  are  formed  ;  and  two  weeks 
after  this  the  ventral  canal-cell  is  cut  off.  Fertilization  takes 
place  five  days  after  the  ventral  canal-cell  ia  formed.  It 
varies  for  the  same  and  different  trees  much  as  pollination 
docs,  so  that  stages  of  fertilization  may  be  obtained  for  a 
week  or  longer.  After  one  season's  experience,  it  is  possible 
to  determine  with  tolerable  accuracy  the  date  of  fertilization, 
but,  after  all,  there  is  an  clement  of  chance  that  can  be 
eliminated  only  by  regular  and  abundant  collections.  I  put 
up  material  from  one  to  three  times  a  day.  The  hour  of 
collection  seerns  to  be  of  little  consequence.  The  central  cell 
was  found  in  active  division  in  cones  collected  at  10  a.m.  in 
bright  sunlight,  in  others  collected  at  7  p.m.,  and  in  still 
others  on  branches  taken  from  the  tree  at  7  p.m.,  and  kept 
in  water  until  11  p.m.  Fertilization  stages  were  abundant  in 
cones  collected  at  9  a.m.  and  at  9  p.m.  of  the  same  day. 

Methods. 

The  material  was  placed  in  the  fixing  solution  within  a  few 
minutes  after  it  was  taken  from  the  tree.  Only  the  middle 
portion  of  the  cone  was  used,  as  this  part  contains  the  best- 
developed  ovules.  In  the  younger  stages,  the  terminal  sterile 
portion  of  the  scale  was  cut  away,  leaving  the  two  ovules 
attached  side  by  side ;  in  older  stages,  the  ovules  were 
entirely  separated  from  the  scale,  and,  as  the  coats  became 
hardened,  they  were  cut  away  at  the  sides,  exposing  the 
endosperm  directly  to  the  fixing  fluid.  The  endosperm 
should  be  quite  well  filled  out  before  this  is  done,  otherwise 
it  may  collapse.  After  the  embryos  are  well  established,  it  is 
well  to  remove  the  coats  entirely.  Many  approved  fixing 
methods  have  been  tried,  with  variations  in  strength,  time, 
and  temperature,  but  for  these  studies  none  has  been  found 
equal  to  Mottier's  modification  of  Flemming's  solution,  used 
fresh  and  allowed  to  act  for  twenty-four  hours  at  about  30°  C. 
During  the  preparation  and  fixing  of  the  ovules  the  bottles 
were  repeatedly   shaken   to  ensure  equal  contact  of  fresh 


t 


^^■«  *--«\'  (J,^'-*^^ 


T 


and  Fertifizalion  in  the  Hemlock  Spruce.       585 

golution  on  all  sides.  The  solution  was  sometimes  changed 
at  the  end  of  one  or  two  hours.  Into  each  bottle  was  placed 
at  the  time  of  fixing  a  small  rectangular  piece  of  linen  paper 
bearing  in  pencil  the  current  number.  This  paper  remained 
with  the  material  through  all  of  the  succeeding  chnnges.appear- 
ing  finally  at  the  bottom  of  the  paraffin  block  as  a  permanent 
and  very  convenient  label.  After  fixing,  the  material  was 
washed  for  twelve  to  twenty-four  hours  in  running  water, 
dehydrated  in  grades  of  alcohol,  bleached  with  a  seventy  per 
cent,  alcoholic  solution  of  hydrogen  peroxide,  and  the 
dehydration  completed  in  commercial  and  absolute  alcohol 
several  times  changed.  It  was  then  brought  very  gradually 
into  cedar  oil,  and  transferred  with  equal  care  into  parailfin 
melting  at  54",  in  which  it  was  imbedded.  I  have  found  it 
expedient  to  store  material  in  seventy  or  eighty  per  cent, 
alcohol  for  a  short  time  after  bleaching,  and  to  allow  a 
number  of  bottles  to  accumulate  before  proceeding  farther 
with  the  imbedding.  Time  and  chemicals  are  thus  saved  and 
more  attention  is  given  to  the  details  of  the  process. 

A  Mmot-Zimmermann  revolving  microtome  was  used  in 
cutting  the  sections.  In  cases  where  the  material  was  poor 
in  stages,  a  number  of  ovules  were  imbedded  in  rectangular 
groups  and  sectioned  together.  Archegonia  were  thus 
examined  by  thousands  instead  of  by  hundreds.  Sections 
were  cut  6*6  ^l  and  1 3-3  (t  in  thickness.  The  ribbons  were 
floated  out  on  water,  fixed  to  the  slide  with  Mayer's  albumen 
fixative,  dried  thoroughly,  and  melted  down  by  placing  the 
slides  in  the  paraffin  bath  for  an  hour  or  two.  The  slides 
were  numbered  with  figures  and  letters  according  to  the 
system  devised  by  Marks.  A  mixture  of  vermilion  and 
sodium  silicate  in  a  little  water  supplied  a  convenient  and 
indelible  medium.  Preliminary  to  staining,  the  slides  were 
passed  through  xylol,  alcohol,  hydrogen  peroxide  solution 
and  water.  Several  staining  combinations  were  used,  the 
well-known  Safranin-Gentian- Violet-Orange  G.  combination 
proving  the  best ;  though  Iron- Hematoxylin  alone  and 
combined  with  acid  Fuchsin,  and  Delafield's  Hematoxylin 


r 


iiaMiiimMwtt^iiw.witiit'**' ;" 


il^ftrf^*f^'*i3'i 


-;i'i-!**at-=--T-^i 


I^  ^  »»^-^^»<iai^!>ii»iMl«'*i'  »*».■,«»  t»-ME'H- 


,«»»>*^s*?*a»'^'^r3^ 


I 
I 


586    Murrill. — TJu  Development  of  the  Archegoniutn 

alone  and  followed  by  Orange  G.  or  Bismarck-Brown,  were 
useful  for  comparison  in  some  stages  of  archegonial  develop- 
ment and  oogenesis.  Methyl-Green,  Fuchsin,  and  Orange  G. 
were  used  in  the  search  for  centrospheres  and  some  slides 
were  covered  without  staining.  After  the  usual  process  of 
dehydration  with  absolute  alcohol  and  clearing  with  oil  of 
cloves,  the  sections  were  treated  for  several  minutes  with 
Bergamot  oil  before  being  covered  with  balsam.  This 
removes  foreign  particles  and  ensures  the  permanence  of 
certain  stains  that  are  extracted  by  the  oil  of  cloves:  it 
also  flows  easily  and  dries  quickly. 

The  Origin  and  Development  of  the  Archegonia 

BEFORE  the  FORMATION  OF  THE  VENTRAL  CANAL-CELL. 

,  The  archegonia  of  Tsuga  arise,  as  they  do  in  other 
Gymnosperms,  from  superficial  cells  at  the  apex  of  the 
prothallium  which  cease  to  divide  and  become  conspicuous 
for  their  large  size  and  the  abundance  of  protoplasm  which 
they  contain.  Fig.  i,  Plate  XXXI,  shows  one  of  these 
archegonial  rudiments  with  its  large  nucleus  and  its  radiating 
bands  of  protoplasm,  which,  in  a  later  stage  (Fig.  a)  are 
confined  chiefly  to  the  upper  portion  of  the  cell,  while  the 
lower  tapering  portion  is  comparatively  empty.  Between 
these  rudiments  are  other  cells  which  continue  to  divide 
(Fig.  3)  and  later  give  rise  to  the  archegonial  sheath  (Fig.  4). 
About  one  week  after  the  differentiation  of  the  archegonial 
rudiment,  the  neck-cell  is  cut  oft"  (Figs.  a-3).  It  at  first  has 
the  form  of  a  circular  disk  surmounting  the  central  cell  and 
equalling  it  in  breadth  but  not  in  height.  As  the  central  cell 
grows,  it  appears  comparatively  narrow  and  more  elongated 
and  also  shows  considerable  variation.  In  the  greater 
number  of  well-developed  archegonfa,  it  divides  into  two  cells 
about  the  time  when  the  ventral  canal-cell  is  formed 
(Figs.  4-6).  The  division-wall  may  be  transverse,  oblique, 
or  longitudinal,  but  it  is  most  often  oblique.  In  many  cases 
the  neck-cell  remains  undivided,  and,  on  the  other  hand,  one 


im 


■ipi|fiii|-|IHMrif»»i-.ni« 


A 


4».< 


and  Fertilization  in  the  Hemlock  Spruce.       587 

frequently  finds  three  or  four  cells  in  the  neck  of  a  mature 
archegonium  (Figs.  8-10).  Differences  of  opinion  concerning 
the  number  of  neck-cells  in  the  archegonia  of  Tstiga  are 
probably  due  to  the  fact  that  the  division  often  occurs  late. 
After  examining  a  large  number,  I  must  agree  with  Hofmeister 
that  two  cells  are  more  frequently  present  than  any  other 
number.  Had  Mottier  (1892)  examined  more  of  the  mature 
archegonia,  he  would  have  probably  found  two  cells  even 
more  frequent  than  he  supposed. 

Returning  to  the  condition  of  the  central  cell  after  the 
neck-cell  was  cut  off,  it  will  be  remembered  that  it  tapers 
towards  its  lower  extremity  and  is  almost  entirely  free  from 
protoplasmic  contents.  Though  its  nucleus  remains  at  the 
apex  just  beneath  the  neck,  rapid  changes  take  place  in  the 
form  and  contents  of  the  cell.  It  increases  greatly  in  size, 
and  a  delicate  reticulum  appears  with  numerous  vacuoles  in 
its  meshes  containing  cell-sap  (Fig.  11).  Enveloping  the 
central  cell  is  a  sheath  of  cells  rich  in  protoplasm  which 
furnish  the  central  cell  with  food,  the  endosperm  still  growing 
vigorously  and  crowding  back  the  disorganized  nucellar 
tissue.  In  place  of  the  delicate  network  shown  in  Fig.  1 1,  the 
central  cell  soon  shows  spherical  vacuoles  filled  with  granules 
and  other  food-masses  (Fig.  12).  These  appear  first  at  the 
periphery  near  the  food-cells  and  later  come  to  occupy  the 
whole  cavity,  with  the  exception  of  one  or  two  large  vacuoles 
at  the  centre.  A  transverse  section  of  a  prothallium  with 
five  archegonia  at  this  stage  is  shown  in  Fig.  16. 

I  cannot  confirm  for  Tsuga  the  results  of  Arnoldi's  recent 
studies  (1900)  on  the  proteid  vacuoles  of  the  Abietineae.  It 
may  be  that  further  search  on  my  part  will  reveal  the  passage 
of  the  nuclei  of  the  sheath-cells  into  the  central  cells,  but 
very  careful  examination  of  numerous  archegonia  in  all  stages 
of  development  has  thus  far  failed  to  show  a  single  un- 
doubted example  of  such  passage.  I  find  the  nuclei  of  the 
sheath-cells  staining  diffusely  at  times,  as  described  by  Ikeno 
(1898)  for  Cycas,  and  I  observe  collections  of  granules  in  the 
outer  vacuoles  of  the  central  cells  which  very  much  resemble 


I  lir Km^miammmimmmmmmtKmHmitKttm 


.  ,..,  j,.,..g..^»u».iaarfiim.mM-i.sB  t 


J 


f ; 


588    Murrill. — The  Da^elopmcnt  of  the  Archegonium 

the  sheath-nuclei,  but  the  sheath-cells  are  never  found  without 
their  nuclei.  The  sheath  remains  one-layered,  though  its 
cells  often  divide  as  the  archegonium  grows.  At  points 
where  the  archegonia  come  into  close  contact  (Fig.  16),  the 
sheath  is  frequently  crushed  and  destroyed,  but  throughout 
most  of  its  extent  the  cells  and  their  nuclei  continue  active 
during  the  life  of  the  archegonium. 

The  Formation  of  the  Ventral  Canal-Cell. 

Shortly  before  the  division  of  the  central  cell,  an  accumu- 
lation of  cytoplasm  may  be  observed  beneath  the  nucleus  a 
little  to  one  side  of  the  longitudinal  axis  of  the  archegonium 
(Fig.  13).  This  accumulation  is  a  dense  mass  of  fibres  with 
small  granules  of  uniform  size  scattered  through  it,  the  whole 
being  continuous  with  the  cytoplasm  around  it  and  taking 
the  same  brownish  stain  with  the  Flemming  combination. 
From  the  first  it  is  closely  pressed  against  the  nuclear  mem- 
brane, and  soon  begins  to  push  it  inward  in  the  form  of  blunt, 
unequal  projections  (Fig.  17).  This  fibrous  mass  continues  to 
increase  in  size  and  to  send  out  radiations  far  into  the  cyto- 
plasm, thus  forming  a  support  for  the  free  lower  pole.  These 
radiations  grow  at  their  free  ends  from  the  cyto-reticulum 
and  extend  in  all  directions,  but  are  of  necessity  short  on  the 
side  next  the  nucleus. 

The  spindle-fibres  arise  within  this  mass  and  grow  upward 
against  and  press  in  the  nuclear  membrane,  while  they  also 
draw  to  a  point  below  and  establish  the  lower  pole  of  the 
spindle  (Figs.  i8-ai).  At  the  pole  there  is  usually  greater 
density  and  frequently  a  rounded  granule  (Fig.  24),  but 
nothing  could  be  discovered  corresponding  to  a  centrosome, 
even  with  the  most  favourable  methods.  Activity  at  the 
upper  pole  begins  late  and  is  always  feeble.  The  cytoplasm 
between  the  nucleus  and  the  neck-rell  is  all  of  a  density 
intermediate  between  that  of  the  ordinary  cytoplasm  and  that 
collected  at  the  lower  pole.  In  a  few  cases  a  minute  hyaline 
lenticular  area  was  observed  at  the  upper  pole,  resembling 
the  collections  of  sap  usually  seen  between  the  polar  caps  of 


«'. 


I 


^ 


•ii 


« 


and  Ferlilization  in  the  Hemlock  Spruce.     589 

dividing  vegetative  nuclei  and  the  nuclear  membrane  (Fig.  15). 
In  later  preparations  this  area  seemed  to  have  broadened  and 
filled  with  delicate  threads  (Fig.  18)  which  later  drew  together 
above,  but  I  cannot  be  sure  of  this  K  Possibly  the  collection 
of  sap  is  so  small  that  it  often  disappears  in  fixing.  Careful 
search  was  made  for  a  similar  collection  at  the  lower  pole, 
but  without  success. 

The  further  development  of  the  spindle  is  very  rapid.  The 
nuclear  membrane  disappears  below  and  the  spindle-fibres 
press  into  the  nuclear  cavity  and  connect  with  the  linen  net- 
work, already  partially  arranged  in  such  a  way  as  to  continue  the 
fibres  to  the  chromosomes  or  through  the  centre  of  the  cavity. 
The  fibres  soon  become  homogeneous  below,  while  at  their 
upper  extremities  they  are  still  only  rows  of  granules  (Fig.  21). 
Nothing  now  remains  in  the  nuclear  cavity  except  the  spindle- 
threads  and  the  homogeneous  chromatin  segments.  The  large 
spreading  bundle  of  fibres  originating  from  below  traverses 
about  two-thirds  of  the  nuclear  cavity  before  connecting  with 
those  from  the  upper  pole.  The  nucleus  consequently  becomes 
pear-shaped  at  this  stage,  with  the  upper  end  larger.  As  the 
spindle-fibres  draw  in  towards  the  centre,  the  chromosomes 
are  forced  from  their  peripheral  position  and  come  to  lie  along 
the  central  part  of  the  spindle  (Fig.  23).  At  the  same  time 
the  upper  pole  is  somewhat  elevated  and  appears  as  an  abrupt 
but  sharp  point  rising  out  of  the  nuclear  cavity.  Supporting 
fibres  run  from  this  pole  to  the  cell-wall  and  in  various 
directions  through  the  neighbouring  cytoplasm  (Fig.  25). 

In  the  plate-stage  (Fig.  26),  the  chromosomes  show  the 
form  and  arrangement  characteristic  of  that  class  of  divisions 
recently  denominated  'typical'  by  Strasburger  (1900),  and 

*  It  is  often  impossible  to  demonstrate  tiie  existence  of  an  extra-nnclear  spindle- 
radinient  at  the  upper  pole,  but  the  fibres  appear  to  collect  in  bundles  within  the 
membrane  and  to  unite  at  one  point  when  the  polarity  becomes  more  pronounced. 
The  narrowing  and  consequent  stretching  of  the  spindle  as  the  metaphase  is 
approached  would  elevate  the  upper  pole  and  give  it  the  appearance  of  penetrating 
the  membrane  (Fig.  22).  In  Larix  Amerkana  there  is  still  less  cytoplasm  above 
the  nucleus  than  in  Tsuga,  which  makes  the  difference  in  the  two  poles  yet  more 
marked.  , 


■MiaaMMMPiilimP 


P^BHi 


rftTf— '"^T*""*"'^'*^*-'*^  ■ '  ■■'  •^'•'  -^ ""'' ' 


I. 


590  A/urri/L — T/ie  Development  of  the    ^rchegonimn 

are  drawn  to  the  poles  as  attenuated  U's  or  V's  (Fig.  27). 
The  cell-plate  is  laid  down  from  the  centre  outwards.  It 
begins  in  the  dispirem  stage  and  keeps  pace  for  some  time 
with  the  growing  daughter-nuclei  (Figs.  29,  30).  The  fibres 
thicken  at  the  centre,  and  the  thickened  portions  fuse  together 
at  the  side.  The  spindle  then  broadens  out  and  stretches  the 
young  cell-plate  until  it  connects  with  the  wall  of  the  mother- 
cell  (Fig.  31).  The  original  nuclear  membrane  does  not 
entirely  disappear  until  the  daughter-nuclei  are  formed.  It 
then  splits  up  into  slender  fibres  and  fades  away  into  the 
surrounding  cytoplasm. 

The  division  above  described,  while  it  resembles  that  de- 
scribed by  Rosen  (1895),  Hof  (1898),  Nemec  (1899),  and 
others  for  the  vegetative*  cells  of  various  plants,  in  the  extra- 
nuclear  origin  of  the  spindle  and  the  details  of  its  construction, 
nevertheless  differs  very  strikingly  from  that  type  by  reason 
of  the  fact  that  the  spindle  is  at  first  asymmetrical  and 
originates  in  a  large  fibrous  mass  beneath  the  nucleus,  which 
appears  early,  grows  to  an  immense  size,  and  remains  for 
some  time  after  the  division  is  completed,  being  enveloped 
during  its  existence  by  numerous  fibres  radiating  from  it  far 
into  the  surrounding  cytoplasm. 

Various  authors  (Ikeno,  1898,  p.  567;  Blackman,  1898; 
Chamberlain,  1899,  figs,  i,  a;  and  others)  have  described  or 
figured  the  nucleus  of  the  central  cell  as  remaining  at  the 
cell-apex  until  division  occurs,  and  it  would  be  interesting 
to  know  whether  more  careful  study  would  show  such  a  method 
of  spindle-formation  as  I  have  discovered  in  Tsuga  in  these 
or  similar  cases  of  unequal  division. 

This  collection  of  cytoplasm  is  probably  due  to  the  need 
of  a  support  for  the  free  lower  pole,  and  the  numerous 
radiations  from  it  doubtless  add  greatly  to  its  stability.  The 
origin  and  growth  of  the  spindle  chiefly  on  the  lower  side 
of  the  nucleus  seem  to  indicate  that  the  force  controlling 
division  is  largely  centred  there. 


1     v 


■MMrt*'-^- 


and  Fcrlilization  in  the  Hemlock  Spruce.     591 


^• 


FuiiTHER  Changes  in  tuE  Egg  and  the  Ventral 

Canal-cell  before  the  Entrance  of  the 

Sperm-cells. 

When  the  nuclear  membrane  is  deposited  about  the  young 
egg-nucleus,  the  chromatin  is  in  the  form  of  a  thick  homo- 
geneous band  which  is  gradually  drawn  out  until  the  chromatin 
granules  appear  distinct  on  the  linin  threads.  At  the  same 
time  the  threads  anastomose  to  form  an  ordinary  cell-reticulum, 
in  which  several  small  nucleoli  appear  (Figs.  29-31).  '^^^ 
nucleus  increases  rapidly  in  size  and  begins  to  move  down 
toward  the  centre  of  the  egg.'  The  chromatin  now  occupies 
chiefly  the  upper  half,  the  lower  part  being  comparatively 
empty  (Fig.  3a).  As  it  begins  to  move,  it  usually  becomes 
elliptical  in  form  (Fig.  33),  and  remains  so  until  fertilized, 
but  if  the  archegonium  is  very  broad  it  may  remain  perfectly 
spherical.  As  it  increases  in  size,  it  takes  in  considerable 
food-material  and  the  network  becomes  coarser  and  the 
nucleoli  larger.  When  the  centre  of  the  egg  is  reached,  it 
remains  stationary  in  the  resting  condition  until  fertilized. 
The  granules  in  the  cytoplasm  of  the  egg  are  arranged  in 
rows  radiating  from  the  nuclear  membrane.  This  arrange- 
ment appears  with  the  formation  of  the  egg  and  disappears 
with  fertilization. 

The  nucleoli  of  the  egg-nucleus  first  appear  as  minute 
spheres  hardly  distinguishable  from  collections  of  chromatin. 
They  increase  rapidly  in  size,  however,  and  apparently  unite 
to  form  larger  ones  and  often  a  single  one  of  immense  size 
(Fig.  35).  When  first  formed,  these  larger  ones  are  very 
plastic  and  easily  take  the  sickle-form  under  the  influence  of 
fixing  agents.  Later,  the  outer  shell  becomes  firm  and  dense 
and  takes  a  deep  purple  stain  with  Gentian-Violet,  while  the 
vesicular  mass  within  stains  feebly  with  Orange^  and  shows 
a  delicate  reticulum  probably  consisting  of  dissolved  proteid 
substance  precipitated  by  chromic  acid.  The  shell  is  frequently 
found  broken  in  mounted  preparations,  and  the  nucleolus  then 

R  r 


I, 


*-«.«K^*'Bf*  <*" 


MMM 


59 2    Murrill. — The  Developmenl  of  the  Archegonium 

suggests  a  sperm-nucleus  with  its  membrane  breaking  away, 
and  the  contents  dissolving  in  the  nuclear  substance  of 
the  egg. 

The  large  vacuole  at  the  centre  of  the  egg  changes  its 
position  as  the  nucleus  moves  down,  and,  passing  it  on  one 
side  (Fig.  34),  comes  to  lie  near  the  apex  of  the  cell.  The 
vacuole  is  apparently  somewhat  diminished  in  size  as  the 
egg-nucleus  increases,  and  it  is  not  improbable  that  some 
of  the  cell-sap  from  the  vacuole  passes  into  the  nucleus  while 
they  lie  near  together.  The  concentration  of  cytoplasm  and 
food  materials  near  the  centre,  in  consequence  of  the  change 
in  position  of  the  nucleus,  leaves  more  room  for  the  vacuole 
near  the  apical  portion  of  the  egg.  Another  vacuole  with 
contents  very  similar  to  those  of  the  nucleus  is  regularly 
observed  *  in  the  mature  egg  situated  just  beneath  the  ventral 
canal-cell  (Fig.  10).  It  is  quite  different  from  the  proteid- 
vacuoles  in  appearance,  but  seems  to  originate  in  one  or  more 
of  the  latter  upon  the  relief  of  pressure  in  the  apical  region 
by  the  enlargement  of  their  enclosing  membranes  and  the 
expansion  and  distribution  of  their  contents.  This  '  nuclear ' 
vacuole  sometimes  fuses  with  the  'empty'  vacuole  as  it 
approaches  the  apex,  but  the  latter  more  often  remains  to  one 
side  and  a  little  below  the  fornier.  Near  these  two  vacuoles 
is  the  receptive  spot  of  the  egg. 

The  ventral  canal-cell  is  fairly  persistent  in  Tsuga.  When 
division  is  completed  its  nucleus  is  equal  in  size  and  similar 
in  structure  to  the  nucleus  of  the  egg,  and  for  some  time 
shows  the  same  stages  of  development.  But  when  the  nucleus 
has  nearly  filled  the  ventral  canal-cell  its  membrane  becomes 
wavy  in  outline,  the  scanty  reticulum  occupies  only  a  small 
portion  of  the  nuclear  cavity,  and  the  nucleoli  remain  minute 
and  scattered.  Growth  having  ceased,  signs  of  disorganization 
soon  appear.  The  nuclear  contents  become  amorphous  and 
stain  diffusely,  while  the  nucleus  and  the  cell  become  more 


fr 


•  An  archegonium  rendered  very  abnormal  by  developing  fcr  down  on  the  «de 
of  the  prothallinm  showed  this  nnclear  Tacuole  in  all  respects  normal. 


turn 

way, 
i    of 

i  its 

one 
The 

the 
ome 
/hile 

and 
inge 
:uole 
with 
larly 
itral 
teid- 
nore 
gion 

the 
lear* 
IS  it 
>  one 
loles 

/hen 
nilar 
time 
:leus 
}mes 
imall 
inute 
lition 
and 
nore 

keside 


j» 


and  Fertilization  in  the  Hemlock  Spruce,     593 

irregular  and  misshapen  in  outline.    It  is  at  this  stage  that 
the  pollen-tube  usually  enters  the  neck  of  the  archegonium. 

The  Entrance  of  the  Contents  of  the  Pollen- 
tube  INTO  THE  Egg. 

The  pollen-tube  reaches  the  egg  by  penetrating  the  neck 
of  the  archegonium,  the  contents  of  the  neck-cells  being 
pressed  to  one  side  or  swept  away  entirely.  They  are  fre- 
quently found  crowded  down  near  the  apex  of  the  egg  with 
the  remains  of  the  ventral  canal-cell  (Fig.  36).  On  reaching 
the  egg  the  tube  spreads  out  over  it  and  causes  its  apex  to 
stain  diffusely.  Further  than  this  the  tube  itself  does  not  go, 
but  the  contents  of  its  terminal  portion  are  emptied  into  the 
egg  near  the  empty  vacuole,  the  membrane  of  the  egg  usually 
closing  up  again,  but  sometimes  remaining  open  at  the  point 
of  entrance. 

The  contents  of  the  pollen-tube  thus  cast  into  the  egg 
consist  of  two  sperm-cells,  the  stalk-cell,  the  vegetative  nucleus, 
and  some  protoplasm  and  starch  from  the  tube-cavity  (Fig. 
36).  The  stalk-cell  nucleus  is  small,  perfectly  spherical,  and 
conspicuous  by  reason  of  its  thick,  deeply-stained  reticulum 
(Fig.  39).  The  cytoplasm  accompanying  it  is  scanty,  vacuolate, 
and  irregular  in  outline.  The  vegetative  nucleus  is  larger, 
ovoid  or  irregular  in  outline,  and  unaccompanied  by  cytoplasm. 
It  contains  a  delicate  network  with  granules  and  nucleoli,  and 
takes  a  pale,  slightly  diffuse  stain  with  Gentian- Violet. 

Above  these  smaller  nuclei  lie  the  ellipsoidal  sperm-cells, 
each  with  dense  cytoplasmic  contents  and  a  large  nucleus.  The 
sperm-nuclei  differ  considerably  in  size  and  appearance.  The 
one  that  was  in  advance  in  the  pollen-tube,  which  I  shall  call 
the  first  sperm-nucleus,  is  about  twice  the  diameter  of  its  com- 
panion, and  its  contents  are  so  dense  that  its  two  small  purple- 
staining  nucleoli  are  almost  hidden  from  view.  The  second 
sperm-nucleus  is  less  dense  and  shows  two  large  and  prominent 
nucleoli  that  stain  a  clear  red  with  safranin.  It  tends  to 
conform  more  to  the  shape  of  its  cell,  and  also  corresponds 
more  nearly  to  the  resting  stage  than  does  the  larger  nucleus. 

R  r  a 


594   Murriil, —  The  Development  of  the  Archegonium 

It  is  through  the  first  sperm-nucleu:i  mat  fertilization  is 
accomph'shed.  A  short  time  after  its  entrance  into  the  egg 
it  slips  from  its  cell  and  moves  with  accelerated  velocity 
towards  the  egg-nucleus,  the  latter  remaining  stationary  and 
inactive,  but  probably  exercising  chemotactic  attraction  on 
the  sperm-nucleus  by  reason  of  its  rich  proteid  contents. 
There  is  no  depression  at  the  apex  of  the  egg-nucleus,  nor 
other  evidence  in  it  of  the  approach  of  the  sperm.  The 
conjugation -path  is  a  straight  line  from  near  the  point  of 
entrance.  Since  this  point  is  ordinarily  at  the  side  near  the 
top  of  the  egg,  the  sperm-nucleus  usually  strikes  the  egg- 
nucleus  slightly  to  one  side  of  its  apex ;  when  entrance  into 
the  egg  is  eflfected  at  the  apex,  the  sperm-nucleus  strikes 
the  egg-nucleus  directly  at  its  apex  (Fig.  38).  Conjugation 
occasionally  occurs  at  the  middle  of  one  side  of  the  egg- 
nucleus,  as  shown  in  Fig.  40.  The  same  figure  also  shows 
the  second  sperm-nucleus  very  near  the  egg-nucleus,  but  there 
is  nothing  in  this  or  other  preparations  of  mine  to  indicate 
that  both  sperm-nuclei  ever  unite  with  the  nucleus  of  the 

egg- 
In  Conifers,  the  sperm-cell  is  very  similar  to  a  pure 
vegetative  cell.  In  the  Pteridophytes,  the  nucleus  is  con- 
densed and  elongated,  with  reduced  cytoplasm  and  active 
cilia.  In  Phanerogams,  the  nuclei  may  be  elongated,  but 
cytoplasm  and  cilia  are  absent.  The  sperm-cells  of  Ginkgo 
and  the  Cycads  diflfer  from  those  of  the  Conifers  in  possessing 
a  cilia-bearing  band  which  propels  the  cells  from  the  tip  of 
the  pollen-tube  to  the  apex  of  the  egg.  In  the  Conifers,  the 
pollen-tube  penetrates  to  the  egg  and  the  cilia-bearing  band 
is  unnecessary  and  absent. 

After  the  first  sperm-nucleus  has  moved  to  the  egg-nucleus 
the  second  sperm-nucleus  remains  for  a  long  time  in  its 
cytoplasm  in  the  upper  part  of  the  egg  (Fig.  38),  and  is  then 
gradually  absorbed,  usually  after  the  other  contents  of  the 
pollen-tube  have  disappeared.  The  discovery  of  a  tripolar 
spindle  (Fig.  46)  in  the  position  commonly  occupied  by  the 
second  sperm-nucleus  at  first  led  me  to  believe  that,  being 


f 


,i '-,- 


and  Fertilization  in  the  Hemlock  Spruce.     595 

fed  by  the  egg-cytoplasm,  this  nucleus  had  entered  upon 
mitotic  division,  just  as  the  functional  sperm-nucleus  pre- 
sumably initiates  the  division  which,  with  the  assistance  of 
the  egg-nucleus,  results  in  the  first  segmentation.  In  the 
nuclei  of  unfertilized  eggs,  also,  the  process  of  disintegration 
may  be  accompanied  by  the  fusion  of  the  chromatic  reticulum 
into  rods  similar  to  chromosomes,  while  the  fibres  of  the 
nuclear  membrane  focus  on  several  points  external  to  the 
membrane  as  the  nuclear  cavity  diminishes,  thus  forming  a 
figure  resembling  a  multipolar  spindle.  Occasionally,  before 
the  nucleus  begins  to  diminish  in  size,  the  disintegrating 
chromatin  contents  are  found  collected  near  together,  with 
numerous  radiations  present  in  the  nuclear  cavity,  the  whole 
suggesting  a  possible  stage  of  fertilization. 


Fertilization  and  the  first  segmentation. 

When  the  egg-nucleus  is  reached,  the  sperm-nucleus  flattens 
itself  against  it  in  the  form  of  a  bi-convex  lens  and  soon 
comes  to  lie  within  its  original  boundary  (Fig.  37).  The 
surface  of  separation  is  at  this  time  quite  even  and  is  com- 
posed of  the  two  nuclear  membranes  with  some  included 
cytoplasm.  The  reticulum  in  the  apex  of  the  egg-nucleus 
is  pressed  down  in  advance  of  the  sperm-nucleus  and  furnishes 
the  first  deposit  of  chromatin  at  that  point. 

The  difference  in  the  density  of  the  sperm-nucleus  and  egg- 
nucleus  is  very  apparent  when  they  are  thus  first  in  contact,  but 
the  former  begins  almost  immediately  to  lose  its  density  and  to 
become  a  perfect  resting  nucleus  like  that  of  the  egg.  In  the 
process,  no  stainable  substance  is  cast  out  into  the  cytoplasm, 
such  as  is  described  by  Ikeno  (1898)  for  Cyeas,  and  Wager 
(1899)  for  some  Phycomycetes  \  nor  can  any  of  its  contents 
be  seen  to  pass  through  the  membranes  into  the  cavity  of 
the  egg-nucleus ;  but  increase  in  the  number  or  in  the  size 
of  the  nucleoli  of  the  sperm-nucleus  is  very  evident  (Figs. 
41^  42).  The  deeply  staining  nucleoli  occupy  the  centre  of 
the  nucleus,  while  its  periphery  shovyrs  a  delicate  chromatic 


.  TijiiirtfifimiiiiiwHiin 


ZSSEES^ 


u 


596   Murnll, — The  Development  of  the  Archegonium 

reticulum,  which  may  have  been  present  before,  concealed 
by  the  dense  contents  of  the  nucleus,  or  may  arise  only  after 
the  contact  of  the  sexual  nuclei.  Certain  granules  of  this 
reticulum  now  gradually  become  larger  by  the  addition  of 
neighbouring  granules,  and  the  whole  contracts  to  a  coarse, 
knotted, slightly  anastomosing  thread,  which,  with  the  assistance 
of  the  nucleoli,  passes  over  into  the  spirem  band  (Figs.  43-45)* 

While  the  sperm-nucleus  has  thus  been  entering  upon  the 
early  stages  of  division,  the  chromatin  of  the  egg-nucleus  has 
been  collecting  near  the  centre  of  the  nuclear  cavity  not  far 
from  the  membranes  separating  the  two  nuclei.  It  likewise 
presents  the  appearance  of  an  anastomosing,  knotted  thread, 
in  contact  with  granules  and  spheres  of  various  sizes  apparently 
derived  from  the  nucleoli,  which  latter  now  become  hollow 
and  stain  feebly,  and  finally  disappear. 

Changes  have  also  occurred  meanwhile  where  the  nuclear 
membranes  are  in  contact.  Instead  of  the  even  surface  pre- 
sented at  the  first  contact  of  the  nuclei,  the  membranes  are 
now  separated  by  numerous  spherical  granular  areas,  which 
tend  to  encroach  on  the  cavity  of  the  egg-nucleus  and  cause 
its  membrane  to  show  in  cross-section  a  series  of  crenate 
folds.  The  contents  of  these  spheres  stain  very  slightly,  with 
the  exception  of  one  or  two  small  spherical  bodies  which  are 
precisely  like  nucleoli  and  take  the  nucleolar  stains.  The  dis- 
appearance of  the  membranes  and  the  consequent  union  of 
the  two  nuclear  cavities  first  occurs  at  points  between  these 
granular  spheres,  and  the  latter  continue  to  occupy  their 
position  until  the  appearance  of  the  spindle-iibres  among 
them,  when  all  of  them  disappear  except  a  few  upon  which 
the  fibres  are  centred.  Whether  they  have  any  direct  con- 
nexion with  the  formation  of  the  spindle,  or  are  simply  cavities 
between  the  nuclear  membranes  containing  a  small  amount 
of  cytoplasm  caught  between  the  conjugating  nuclei,  it  is 
impossible  for  mc  to  say.  Their  increase  in  size  with  the 
decrease  in  density  of  the  sperm-nucleus  has  suggested  to  me 
the  arrangement  found  in  Cycas  and  Cephalotaxus,  where 
fusion  is  accelerated  by  root-like  projections  of  the  sperm- 


aMMM 


mum 


and  Fertilization  in  the  Hemlock  Spruce.     597 

nucleus  into  the  egg-nucleus ;  but,  even  if  the  analogy  were 
otherwise  perfect,  the  increase  in  the  amount  of  nucleolar 
substance  seems  to  fully  account  for  all  the  contents  of  the 
sperm-nucleus  not  found  in  its  chromatic  reticulum. 

The  breaking  down  of  the  membranes  separating  the  two 
nuclear  cavities  occurs  before  the  completion  of  the  spirem 
bands,  but  the  chromatin  masses  remain  distinct  until  the 
chromatic  segments  appear.  The  achromatic  contents  of 
the  cavity,  however,  undergo  a  decided  change,  becoming 
denser  and  more  fibrous  in  appearance  with  the  probable 
rearrangement  of  the  linin  network  under  the  stimulus  causing 
division  (Fig.  45).  The  outer  membrane  also  disappears  in 
places,  and  some  of  the  cytoplasm  presses  into  the  cavity,  but 
the  chief  activities  of  division  are  intranuclear.  With  the 
union  of  the  nuclear  cavities  there  also  occurs  a  change  in 
the  cytoplasm  of  the  egg.  The  dense  sheath  of  small  granules 
and  fibres  that  encircles  the  unfertilized  egg-nucleus  partly 
disappears,  and  the  larger  cytoplasmic  granules  extend  almost 
to  the  nuclear  cavity,  while  the  rows  of  elongated  granules, 
radiating  into  the  surrounding  cytoplasm  from  the  egg-nucleus 
since  its  origin,  now  lose  their  radial  position  and  are  dis- 
tributed without  special  arrangement.  The  indications  are 
that  the  egg-nucleus  has  relaxed  its  hold  for  a  time  upon  its 
cytoplasm  to  enter  upon  the  changes  involved  in  division. 

The  fibres  originating  the  spindle  of  the  first  segmentation 
arise  among  the  segmenting  spirems  derived  from  the  two 
nuclei,  and  they  first  draw  together  at  several  different  points 
forming  a  multipolar  spindle-rudiment.  On  these  fibres  the 
long  bent  and  twisted  chromosomes  appear,  still  showing 
the  chromatin  disks  distinct  on  the  linin  thread  (Fig.  47). 
As  the  number  of  fibres  increase,  and  the  spindle  becomes 
monaxial  in  form,  the  chromosomes  contract  and  become 
homogeneous,  and  are  mostly  bent  in  the  form  of  a  U. 
There  is  no  difference  to  be  observed  between  the  chromo- 
somes of  the  sperm  and  those  of  the  egg,  and,  at  this  stage, 
they  are  mingled  indiscriminately  near  the  centre  of  the 
common  spindle. 


MM 


V. 


t 


598    Murrill, —  The  Development  of  the  Archegonium 

The  mature  spindle  is  broad,  with  many  fibres  and  rather 
blunt  ends,  and,  during  the  metaphase,  the  twenty-four  chromo- 
somes occupy  all  of  the  equatorial  plane  (Figs.  50,  51).  The 
type  of  this  division,  so  far  as  the  chromatin  is  concerned,  is 
the  same  as  that  already  described  for  the  central  cell  of  the 
archegonium.  The  position  of  the  mature  spindle  in  the  nuclear 
cavity  seems  independent  of  the  relative  position  of  the  con- 
jugating nuclei,  and,  while  the  division  is  usually  oblique,  it 
may  be  perpendicular  to,  or  parallel  with,  the  longitudinal 
axis  of  the  archegonium  (F"igs.  5a,  54,  57). 

The  chromosomes  pass  to  the  poles  as  slender  undulated 
V's  or  U's,  and  fuse  to  form  the  network  of  the  daughter- 
nuclei  (Figs.  53,  54).  No  cell-plate  is  formed,  but  the  fibres 
fade  away  into  the  cytoplasm,  the  slightly  thickened  middle 
portions  being  the  last  to  disappear. 

The  pro-embryo. 

The  two  free  nuclei  resulting  from  the  first  segmentation 
increase  rapidly  in  diameter,  and  soon  divide  simultaneously 
without  change  of  position  to  form  four  nuclei  of  equal  size 
lying  free  near  the  centre  of  the  egg  (Figs.  54-58).  The  type 
of  the  second  division  is  similar  to  that  of  the  first,  but  the 
spindle  is  narrower  and  more  pointed  at  the  ends  (Fig.  56). 
The  daughter-nuclei  are  formed  and  the  spindle-fibres  absorbed 
as  in  the  first  division.  When  the  four  nuclei  have  attained 
their  full  size,  they  move  to  the  base  of  the  archegonium 
(Figs.  59, 60),  and  by  successive  divisions  in  a  horizontal  plane 
give  rise  to  four  tiers  of  nuclei  with  four  nuclei  in  each  tier, 
as  has  been  already  described  for  various  Conifers. 

When  the  four  free  nuclei  are  moving  through  the  egg- 
cytoplasm  they  do  not  show  any  special  collections  of  fibres 
about  their  membranes^  but  soon  after  the  base  is  reached 
they  become  enclosed  in  a  dense  mass  of  fibrous  substance 
(Fig.  60),  which  supplies  the  material  for  the  walls  that  appear 
later.  Above  this  fibrous  mass  is  a  zone  showing  a  delicate 
reticulum  almost  devoid  of  stainable  substance.    Above  this 


OMMH 


•tF 


nitn 

ther 
mo- 
The 

d,  is 
the 

:lear 
con- 

e,  it 
linal 


and  Fertilization  in  the  Hemlock  Spruce.     599 

zone  the  egg  shows  its  normal  structure.  The  cause  of  this 
h^  aline  zone  is  difficult  to  determine.  It  appears  that  the 
stored  food  has  been  taken  from  this  region  in  order  to 
provide  for  the  growing  want  of  supplies  for  the  nuclei  below  ; 
but  since  the  remaining  available  contents  of  the  egg  are 
doubtless  also  transferred  to  the  developing  embryo,  there  is 
no  apparent  reason  why  this  zone  should  be  left.  It  possibly 
represents  an  intermediate  condition  of  these  contents  in  which 
they  do  not  easily  take  or  retain  stains. 


ated 
iter- 
bres 
ddle 


it  ion 
usly 
size 
type 
the 
56). 
rbed 
ined 
lium 
lane 
tier, 

egg- 
bres 
:hed 
ance 
pear 
icate 
this 


Summary. 

The  archegonia  of  Tsuga  originate  as  single  superficial 
cells,  in  each  of  which  occurs  the  usual  division  cutting  off 
an  outer  smaller  cell  that  forms  the  neck.  At  maturity,  the 
neck  most  commonly  consists  of  two  cells,  though  in  very 
many  cases  the  neck-cell  fails  to  divide  at  all.  Three  and 
four  cells  in  the  neck  are  less  commonly  observed. 

In  the  division  of  the  central  cell  the  spindle-fibres  arise 
from  a  large  dense  fibrous  mass  beneath  the  nucleus  and  grow 
into  the  nuclear  cavity,  where  they  are  later  joined  by  fibres 
from  the  very  small  upper  pole.  The  division  resembles  that 
recently  described  for  many  vegetative  cells,  but  belongs  to 
a  new  and  distinct  type  hitherto  undescribed.  The  mass  of 
cytoplasm  at  the  lower  pole  with  its  extensive  radiations 
suggests  a  huge  centrosphere. 

As  the  egg-nucleus  increases  in  size  and  moves  to  the  centre 
of  the  egg,  the  vacuole  moves  upward,  passing  it  on  its  way 
and  comes  to  lie  near  the  egg-apex.  Just  beneath  the  ventral 
canal-cell  may  always  be  found  another  somewhat  smaller 
vacuole  with  contents  similar  to  those  of  the  nucleus.  These 
vacuoles  form  the  receptive  spot  of  the  egg. 

The  contents  of  the  pollen-tube  cast  into  the  egg  consist 
of  the  vegetative  nucleus  with  some  cytoplasm  and  starch- 
grains,  the  stalk-nucleus  surrounded  by  its  own  scanty,  vacuo- 
late cytoplasm,  and  the  two  unequal  sperm-cells  with  their 
dense  cytoplasm  and  large  deeply-stained  nuclei.     The  larger 


HlMlillT'    " 


itfi^iriiii 


li  1  nfil  iBTJMWfcaiWiW 


, 


6oo  Murrill. — The  Development  of  the  Archegonium 

sperm-nucleus  slips  from  the  cell  and  conjugates  with  the 
egg-nucleus,  the  smaller  one  being  gradually  absorbed  with 
the  Ot'ier  structures  derived  from  the  pollen-tube. 

The  lunctional  sperm-nucleus  flattens  itself  against  the 
egg-nucleus  in  the  form  of  a  bi-convex  lens,  and  the  two 
nuclei  soon  come  to  lie  in  the  space  formerly  occupied  by  the 
egg-nucleus  alone,  their  membranes,  however,  remaining  intact 
for  a  long  time.  In  this  condition  the  sperm-nucleus  rapidly 
loses  its  density  and  constructs  a  delicate  peripheral  chromatic 
reticulum  and  larger  central  nucleoli,  thus  becoming  a  perfect 
resting  nucleus  similar  to  that  of  the  egg.  The  chromatin  of 
each  nucleus  collects  in  the  form  of  a  thick  knotted  thread 
near  the  centre  of  the  separating  partition,  and  the  two  masses 
remain  distinct  until  the  spirem-bands  begin  to  segment. 

Just  before  the  spirems  are  formed  the  separating  mem- 
branes disappear  and  the  nuclear  cavities  become  united. 
The  spindle  then  arises  in  a  multipolar  fashion  between  and 
among  the  two  masses,  twelve  chromosomes  being  supplied 
from  the  chromatin  of  the  sperm  and  twelve  from  that  of  the 
egg,  as  described  by  Blackman  for  Pinus  Sylvestris.  The 
mature  spindle  is  broad  with  blunt  ends,  and  the  manner  of 
division  is  typical. 

A  second  division  succeeds  the  first  without  much  delay, 
and  the  four  resulting  free  nuclei  soon  attain  full  size  and 
move  to  the  base  of  the  arch^onium,  where  the  young  embryo 
becomes  established  in  the  manner  already  so  well  known 
among  Conifers. 

The  investigations  leading  to  the  results  recorded  above  were 
conducted  in  the  Botanical  Laboratory  of  Cornell  University 
under  the  direction  of  Professor  Geoi^e  F.  Atkinson,  at  whose 
suggestion  this  work  was  undertaken,  and  for  whose  kindly 
sympathy  and  invaluable  aid  in  its  prosecution  I  aqi  deeply 
grateful. 


Vt 


wilBiiiiiini 


.isgi'^ 


and  Fertilization  in  the  Hembck  Spruce.     60 1 


Bibliography. 

Andrews,  G.  F.,  '98  :  Camera  Drawing.    Zeitschr.  f.  wist.  Mikr.,  xiv,  451-45J, 

1898. 
Aknoldi,  W.,  '00' :   Beitrage  zur  Morphologic  der  Gymnoipermen :   iii.    Flora, 

Ixxxvii,  46-63,  pi.  1-3, 1900. 
'OO'' :  Beitrage  zur  Morphologic  der  Gymnoapermen :  iv.    Flora, 

Ixxxvii,  194-204,  pi.  6,  1900. 
Blackman,  V.  H.,  '98  :  On  the  Cytological  Features  of  Fertilization  and  Related 

Phenomena  in  Pinus  Sylvestris,  L.    Phil.  Trans.  Roy.  Soc,   B.,  cxc, 

395-426,  pi.  IJ-14,  1898. 
Chamberlain,  C.  J.,  '99  :  Oogenesis  in  Pinus  I.aricio,  Poir.    Bot.  Gaz.,  xxvii, 

268-280,  pi.  4-6,  1899. 
Dixon,  H.  H.,  '99 :  The  Possible  Function  of  the  Nnclcolus  in  Heredity.    Ann. 

Bot.,  xiii,  269-278,  1899. 
FULMER,  E.  L.,  '98:  Cell  Division  in  Pine  Seedlings.    Bot.  Gaz.,  xxvi,  239-246, 

pi.  33,  34, 1898. 
GuiGNAKD,  L.,  '91 :   Nouvelles  Etudes  snr  la  F^condation.    Ann.  d.  so.  nat., 

Botaoiqne,  7*  s^r.,  t.  xiv,  163-296,  pi.  9-18,  1891. 
HoF,  A.  C,  '98 :  Histologiache  Studien  an  Vegetationspnnkten.    Bot.  Centralbl., 

Ixxvi,  Nos.  3-7,  pi.  3-4,  1898. 
Ikeno,  S.,  '98:  Untersuchnngen  iiber  die  Entwickelnog  der  Geschlechtsorgane 

und  den  Vorgang  der  Befmchtnng  bei  Cycas  revoluta.    Jahr.  f.  wiss. 

Bot.,  xxxii,  557-602,  pi.  8-10,  1898. 
MOTTIER,  D.  M.,  '92 :  On  the  Archegoninm  and  Apical  Growth  of  the  Stem  in 

Tsuga  Canadensis  and  Pinus  Sylvestris.   Bot.  Gaz.,  xvii,  141-143,  pi.  8, 

1892. 
Nehec,  B.,  '98  :  Ueber  die  Ausbildnng  der  achromatischen  Kemtbeilungsfigur 

im  vegetativen  und  Fortpflanzungsgewcbe  der  hoheren  Pflanzen.    Bot. 

Centralbl.,  Ixxiv,  1-4,  8  text-figures,  1898. 
'99' :  Ueber  Kern-  und  Zelltheilnng  bei  Solanum  tubtrosum.    Flora, 

Ixxxvi,  314-237,  pi.  13-14,  1899. 

'99* :  Ueber  die  karyokinetische  Kemtheilung  in  der  Wnrzelspitze  von 


Allium  Cepa.    Jahr.  f.  wiss.  Bot.,  xxxiii,  313-336,  pi.  3,  1899. 
Rosen,  F.,  '95  :  Kerne  und  Kemkorperchen  in  meristematischen  und  sporogenen 

Geweben.    Cohn's  Beitr.  z.  Biol.  d.  Pflanc,  vii,  335-313,  pi.  3-4,  1895. 
Strasburger,  £.,  '84  :  Ncne  Untersuchnngen  iiber  die  Be&uchtungsvorgange  bei 

den  Phanerogamen  als  Grundlage  fUr  eine  Theorie  der  Zeugnng.   Jena, 

1884. 
'88 :  Ueber  Kern-  und  Zelltheilung  im  Pflanzenreiche,  nebat 

einem  Anhang  iiber  Befruchiung.    Histol.  Beitr.,  i,  1888. 

'92 :  Ueber  das  Verhalten  des  Pollens  und  die  Befruchtungi- 


vorgiinge  bei  den  Gymnospermen.    Histol.  Beitr.,  iv,  1893. 

*97' :  Kemtheilung  und  Befruchtung  bei  Fucus.    Jahr.  f.  wiss. 


Bot.,  XXX,  35'-374.  pl-  17-18,  1897. 

'97*:  Ueber  Befruchtung.    Jahr.  f.  wi»s.  Bot.,  xxx,  406-422, 


1897. 


-A^ 


6o2   Mtirrill. —  The  Development  of  t lie  Archegonium 

Strasbukuer,  K.,  '00:  Ueber  Reductionstheilung,  Spindel'  Mung,  Centrosomen, 

und  Cilienbiklner  im  Pflanzenreich.    Jena,  1900. 
Thom,  C,  '99  :  The  Process  of  Fertilization  in  Aspidium  and  Adiantum.    Trans. 

Acad.  Sci.  St.  Lonis,  ix,  385-314^  pi.  36-38,  1899. 
Wager,  H.,  '99  :  The  Sexuality  of  the  Fungi.    Ann.  Bot.,  xiii,  575-597,  1899. 
Wai.deyer,  W.,  '88 :  Ueber  Karyokinese  und  ihre  Beziehung  zu  den  Befruchtnngs- 

vorgiingen.    Arch.  f.  milcr.  Anat.,  xxxii,  i -12a.     14  text-figures.    1888. 


EXPLANATION   OF  FIGURES   IN   PLATES 
XXXI  AND   XXXII. 

Illustrating  Dr.  Murrill's  paper  on  Tsuffi  Canadensis. 

Most  of  the  figures  were  first  drawn  with  the  aid  of  a  camera  lucida  from  a  Zeiss 
microscope,  using  compensation  ocular  1 3  and  the  a""  homogeneous  objective,  and 
then  mechanically  reduced  to  the  present  scale.  For  some  it  was  necessary  to  use 
a  lower  magnification  in  the  first  instance.  In  using  the  camera  lucida,  I  found  it 
convenient  to  employ  dark  shades  of  paper  (Andrews,  1898),  usually  black  or  dark 
blue,  and  the  outlines  were  traced  upon  it  in  Chinese  white  with  a  long  pointed 
pen.  Since  most  of  the  preparations  are  coloured  with  dark  stains,  a  white  medium 
is  peculiarly  suitable  for  outlining  chromatic  structures. 

PL.VFE  XXXI. 

Fig.  I.  (  X  400.)  A  portion  of  the  apex  of  the  prothallium,  showing  an  arche- 
gonial  rudiment  in  the  centre,  with  rudimentary  sheath-cells  on  each  side. 

Fig.  3.  (x  400.)  The  archegonial  rudiment  has  increased  in  length,  and  iti 
nucleus  is  preparing  to  divide.  Almost  all  the  protoplasm  is  collected  at  the  upper 
end  of  the  cell    The  sheath-cells  have  increased  in  number. 

Fig.  3-  ( X  400.)  The  archegonial  rudiment  has  divided  into  an  inner  central 
cell  and  an  outer  neck-cell.    The  outer  cell  is  much  smaller  than  the  inner. 

Fig.  4.  ( X  300.)  The  neck-cell  is  in  division.  The  central  cell  is  very  much 
larger,  and  is  being  rapidly  filled  with  protoplasm  from  the  rich  layer  of  sheath- 
cells  that  closely  envelop  it.  Its  nucleus  remains  near  the  neck-oell  and  is  still  in 
the  resting  stage,  though  the  condensation  of  cytoplasm  just  below  it  indicates  that 
division  is  not  far  off. 

Fig.  5.  (  X  400.)  A  later  stage  of  the  same,  showing  the  anaphase  of  the 
division  in  the  neck-cell. 

Fig.  6.  ( X  400.)  A  still  later  stage,  showing  the  neck-cell  divided  into  two 
cells.    The  central  cell  is  now  entering  upon  division. 

Fig.  7.  ( X  300.)  A  mature  archegonium,  with  two  cells  in  the  neck.  The 
division  wall  is  oblique.     The  ventral  canal-cell  is  also  shown. 


\ 


\um 


and  Fertilization  in  the  Hemlock  Spruce.     60j 


>men, 
'r«ns. 

99- 

Bng«- 

888. 


Zeiss 
I,  and 

O  DSC 

indit 
dark 
linted 
dium 


jche- 

id  it! 
ipper 

»tral 

much 
eath- 
lill  in 
I  that 

f  the 

>  two 

Th« 


\ 


Fig.  8.  (  y  300.)  A  matnre  arcbegonium,  with  three  cells  in  the  neck.  The 
iirst  division  was  transverse,  and  the  upper  cell  afterwards  divided  longitudinally. 

Fig.  9.  ( X  aoo.)  Four  neck-cells  are  present,  formed  by  one  longitudinal 
division  followed  by  two  oblique  ones.  The  cytoplasm  of  both  cells  has  divided, 
bnt  no  separating  walls  could  be  observed. 

Fig.  10.  (x  aoo.)  In  this  arcbegonium  the  four  neck-cells  are  in  one  row, 
formed  by  two  successive  transverse  divisions.  Beneath  the  neck  is  the  dis< 
organizing  ventral  canal-cell,  and  beneath  the  ventral  canal-cell,  at  the  apex  of  the 
egg,  is  the  nuclear  vacuole.  The  empty  vacuole  is  in  another  section.  At  the 
centre  of  the  egg  is  the  large  resttiig  nucleus.  About  the  periphery  of  the  egg, 
proteid  vacuoles  are  abundant.  The  larger  granules  of  cytoplasm  are  arranged  in 
rows  radiating  from  the  nucleus.  The  contents  of  the  egg  have  become  so  dense 
that  its  reticulum  is  wellnigh  concealed.  In  many  places,  also,  the  reticulum  has 
been  distorted  by  denser  aggregations  of  nutritive  materials. 

Fig.  II.  (  X  aoo.)  This  follows  Fig.  3  in  the  order  of  development,  bnt  shows 
a  marked  increase  in  the  size  of  the  central  cell,  with  a  well-formed  sheath.  The 
nucleus  is  at  the  apex  of  the  cell,  where  it  remains  until  division.  Throughout  the 
cell-cavity  a  delicate  reticulum  has  been  constructed,  which  is  interrupted  at  the 
centre  of  the  cell  by  a  large  vacuole  filled  with  sap. 

Fig.  I  a.  (x  aoo.)  A  later  stage  in  the  development  of  the  arcbegonium.  The 
neck-cell  has  elongated.  The  central  cell  has  enlarged,  and  the  meshes  of  its 
rnticulum  are  fast  filling  with  granular  food  supplies.  The  first  proteid-vacnoles 
have  been  formed. 

Fig.  13.  (  x  400r)  This  figure  represents  the  nucleus  of  the  central  cell  pre- 
paring to  divide.  Its  reticulum  has  become  coarser  and  stams  more  deeply,  and 
is  balled  up  in  the  cedtre  of  the  nuclear  cavity  in  a  condition  suggesting  synapsis. 
The  preparations  showing  this  condition  are  fixed  as  perfectly  as  one  could  desire. 
Beneath  the  nucleus  is  a  dense  fibrous  mass  closely  pressed  against  the  nuclear 
membrane,  and  sending  out  radiating  fibres  into  the  cytoplasm. 

Fig.  14.  (  X  Soo.')  The  spindle-fibres  are  arising  in  this  mass,  and  growing 
upward  against  the  membrane. 

Fig.  15.  (x  800.)  A  lenticular  hyaline  area  at  the  upper  pole,  seen  in  only 
two  or  three  preparations,  but  very  distinct  and  apparently  perfectly  normal. 

Fig.  16.  (x  150.)  A  cross-section  of  a  prothallium  containing  five  archegonia 
of  the  stage  shown  in  Fig.  la.  It  will  be  observed  that  each  archegonium  is 
enveloped  by  its  own  one-layered  sheath.  The  archegonia  are,  therefore,  ordinarily 
separated  by  two  rows  of  sheath-cells,  but  at  points  where  there  is  some  distance 
between  the  archegonia  the  cells  of  the  two  sheaths  have  divided  to  fill  the  space. 
Between  the  two  upper  archegonia  in  the  figure,  the  sheaths  have  been  crushed  to 
a  line.  This  is  quite  frequently  (he  case  where  the  curved  surfaces  of  the  arche- 
gonia come  nearest  together. 

Fig.  17.  (x  800.)  Following  Fig^  14,  and  showing  the  pressing  in  of  the 
nuclear  membrane,  in  the  form  of  blunt  protuberances,  by  the  spindle-fibres  origi- 
nating below.  The  chromatin  thread  is  now  peripheral  and  almost  continuous, 
though  the  disks  are  still  far  apart. 

Fig.  18.  ( X  800.)  The  spindle-fibres  have  advanced  still  farther,  and  pre- 
parations are  being  made  within  the  nuclear  cavity  for  their  continuation. 
Segmentation  occnn  abont  this  time,  and  rows  of  granules  and  delicate  threads 
connect  the  chromatic  segmeati  with  the  incoming  spindle-fibres.    The  greater 


|]w"iiili' 


I 

1%- 


604  Murrill. —  The  Development  of  the  Archegonium 

part  of  the  chromatin  is  in  the  upper  half  of  the  nncleui.  Activity  has  now  begun 
at  the  upper  pole,  where  delicate  fibres  are  seen  growing  down  a^^nst  the  nuclear 
membrane,  but  it  rarely  appears  so  distinct  as  m  this  preparation. 

Fig.  19.  ( X  800.)  This  nucleus  belongs  to  a  larger  archegonium  than  that 
shown  in  Fig.  18.  The  spindle  is  in  about  the  same  stage,  but  the  chromosomes 
are  more  advanced  than  those  shown  in  the  preceding  figure.  Radiations  are 
present  at  the  upper  pole,  and  possibly  a  small  polar  cap,  but  the  latter,  if  present, 
is  not  distinct  enough  to  figure. 

Fig.  20.  ( X  800.)  This  figure  represents  the  same  stage  as  Fig.  19,  but  the 
nucleus  remains  spherical,  and  the  spindle-fibres  seem  to  have  originated  at 
a  gteater  distance  than  usual  below  the  nuclear  membrane,  and  to  have  advanced 
with  a  more  even  front.  This  u  exceptional,  being  observed  only  a  few  times  in 
the  examination  of  a  large  number  of  preparations. 

Fig.  ai.  ( X  800.)  The  nuclear  membrane  has  disappeared  opposite  the  polea, 
and  the  spreading  cone^haped  bandies  of  spindle-fibres  have  grown  into  the 
nuclear  cavity,  and  are  uniting  somewhat  above  the  centre  of  the  nucleus.  The 
true  pear-shaped  form  of  the  nucleus  and  the  inequality  of  the  two  poles  cannot  be 
shown  in  a  longitudinal  lection  Which  includes  the  upper  pole,  smoe  the  division  is 
oblique  and  much  of  the  lower  pole  is  cut  away.  When  first  formed,  the  spindle 
is  broad  and  the  chromosomes,  which  have  now  become  homogeneona,  are  attached 
to  its  outer  threads. 

Fig.  23.  ( X  800.)  The  spindle-fibres  are  now  homogeneous  throughout,  and 
the  spindle  has  narrowed  and  drawn  in  the  completed  chromosomes  neater  to  the 
centre.  In  narrowing,  it  has  also  elongated,  and  the  upper  pole  has  been  elevated. 
The  section  is  made  as  in  Fig.  ai,  and  does  not  show  all  of  the  lower  pole. 

Fig.  23.  (  X  800.)  Further  narrowing  of  the  spindle  has  taken  place,  and  the 
chromosomes  are  now  coming  up  to  the  equator,  preparing  to  enter  the  plate  stage. 
The  equatorial  portion  of  the  nucleus  approaches  very  near  the  wall  of  the  egg, 
and  above  this  line  the  small  cap  of  cytoplasm  is  dense  and  full  of  fibres. 

Fig.  24.  (  X  400.)  The  lower  pole  of  the  mature  spindle  is  here  represented. 
The  fibres  come  to  a  definite  point,  and,  in  this  case,  focus  on  a  small  hyaline 
granule,  which  shows  none  of  the  properties  of  a  oentrosome. 

Fig.  25.  (  X  400.)  The  fibres  of  the  mature  spindle  also  ccnverge  to  a  definite, 
though  rather  abrupt,  pomt  at  the  upper  pole,  from  which  supporting  fibres  extend 
to  the  cell-wall.  The  remainder  of  the  nuclear  membrane  also  appears  to  function 
as  a  support  to  the  spindle  during  metakinesis. 

Fig.  26.  ( X  800.)  Separation  of  the  chromosomes  has  begun  at  the  nuclear 
plate,  the  bundles  of  nutntle-fibres  being  attadted  on  opposite  sides  of  the  diamond- 
shaped  openings  in  the  chromosomes  already  seen  in  earlier  stages. 

Figs.  27,  28.  (  X  600.)  The  daughter-chromosomes  pass  to  the  poles  as  U's  or 
V's  with  undulated  margins.  After  they  reach  the  pnles,  the  central  apindle-fibras 
appear  lax,  and  the  spindle  becomes  slightly  concave  in  the  equatorial  region. 
This  may  be  due  to  artificial  cause*  or  to  relaxation  after  removal  of  the  strain. 

Fig.  39.  (X400.)  The  daughter-chromosomes  have  united  into  a  close,  dee[dy> 
stainhig  spirem,  but  no  membrane  is  yet  formed  about  theiA.  The  inner  spindle* 
fibres  are  beginning  to  thicken  in  the  equatorial  region  preparatory  to  the  formation 
of  the  cell-plate. 

Fig.  30.  (x  400.)  The  diqiirems  have  opened  ont,  bat  their  loops  still 
maintain  a  position  parallel  with  the  axis  of  the  spindle.    Delicate  nuclear 


^ 


»(«#'* 


mm 

begun 
iclear 

I  that 
tomet 
It  are 
nent. 

It  the 
ed  at 
anced 
let  in 

poles, 
a  the 
The 
lotbe 
ion  it 
>indle 
iched 

,  and 
:othe 
rated. 

dthe 
itage. 

nted. 
«line 

inite, 
(tend 
ction 

dear 
lond- 

Vtat 
Ums 
gion. 
1. 

ndle* 
ttion 

ttiil 


1 


MMMI-' 


and  Ferltlizalion  in  the  Hemlock  Spruce,     605 

bianet  have  been  depoiited  abont  the  danghter-nuclei,  while  the  latt  tracet  of  the 
membrane  of  the  mother-nucleut  have  disappeared.  The  large  dente  matt  of 
cytoplaim  at  the  lower  pole,  however,  still  remaint,  and  is  present  even  when  the 
egg-nncleni  begint  to  past  down.  The  formation  of  the  cell-plate  it  proceeding 
fori  passu  with  the  increase  in  diameter  of  the  daughter-nuclei. 

Fig.  31.  (x  400.)  The  danghter-nuclei  have  reached  the  resting  stage,  and 
thow  a  delicate  reticnlnm  with  teveral  nucleoli.  The  egg-nucleus  it  somewhat 
larger  than  that  of  the  ventral  canal-cell.  The  cell-plate  hat  been  continued  to 
the  wall  of  the  mother-cell. 

PiK-  3'-  ( ><  400.)  The  egg-nncleut  it  now  not  only  larger  than  the  ventral 
canaUcell  nucleus,  but  itt  reticulum  is  growing  more  rapidly.  The  chromatic 
contents  of  the  nucleus  are  mottly  confined  to  itt  upper  portion,  the  lower  part 
containing  chiefly  nuclear  sap. 

P'S-  .13-  ( '*'■  4°oO  "^^  reticulum  of  the  egg-nncleus  has  become  coarser 
and  the  nucleoli  larger.  The  nucleus  has  alto  changed  itt  form  from  ipherical  to 
elliptoidal,  and  bat  begun  to  move  through  the  dente  polar  cytoplatm  toward 
the  centre  of  the  egg.  The  larger  granules  of  the  geneml  cytoplasm  are 
arranged  in  radial  rows  abont  the  nucleus,  and  the  grannies  themselvet  are  radially 
elongated. 

F'S-  34-  (  X  >5^)  At  ^i>  *'*8e  ^'^  reticulum  of  the  egg  nncleui  it  distributed 
throughout  the  entire  nuclear  cavity.  At  the  archegonium  it  nnutually  broad,  the 
nucleut  hat  retained  iu  spherical  form.  Passing  it  on  the  left  is  the  large  central 
vacuole  which  now  takes  a  position  near  the  egg  apex.  The  nucleus  of  the  ventral 
canal  cell  it  irregular  in  outline,  and  ita  contents  thow  tignt  of  disorganization. 
Tracet  of  the  divition-tpindle  ttill  remain  above  the  nucleus. 

PLATE  XXXII. 

Fig.  35-  (  X  300.)  The  nucleus  here  represented  it  drawn  from  an  egg  into 
which  the  contentt  of  the  pollen-lube  have  just  been  diacharged.  Situated  near 
the  bate  of  the  nucleus,  it  the  very  large  nucleolui  with  its  firm,  deeply  ttaining 
outer  thell  broken  at  one  point.  The  contenU  of  the  nudeolut  appear  finely 
giannlar  and  vacuolate  in  ttained  preparations. 

Fig-  3<i'  (^  ><>°0  "I^  contentt  of  the  pollen-tube  have  entered  the  egg 
near  itt  apex  on  the  right  hand  tide  in  the  figure,  and  now  lie  beneath  the  empty 
vacuole  with  the  functional  tperm-nndeut  in  advance,  and  already  free  from  iU 
cytoplatm.  Near  it  on  the  left  it  the  ttalk-cell,  while  between  it  and  the  tperm- 
ocU  Uct  the  vegetative  nucleut.  The  contentt  of  the  firtt  tperm-nucleut  are  very 
dense  and  stahi  deeply.  The  egg-nndeut  it  apparently  unaffected  by  the  near 
approach  of  the  tperm.  Jutt  above  the  %g  are  the  remaint  of  the  neck-cellt  and 
the  ventral  canal-cell  puhed  atide  by  the  entering  pollen-tube. 

Fiff-  37'  (  ^  '^0  '^'^  ^^  tpeim-nnclent  has  flattened  itielf  against  the 
apex  of  the  egg-nndeus  in  the  form  of  a  biconvex  lent.  Its  contentt  have  at  yet 
undergone  no  change.  The  tecood  iperm-nnclens  remaint  in  itt  cytoplatm  above, 
ihowtaig  itt  ictkmlam  and  nucleoli  very  dittinctly.  The  tmaller  nuclei  are  in 
another  tection.  The  first  speim-audcnt  hat  left  slight  traces  of  iU  passage  to  the 
cge^nudens  ia  the  intervening  cytoplasm.    These  traces  are  mwe  distinct  in 

Fig.  38. 
Fig.  38.    (x  200.)    The  sperm-nudeus  hat  lott  itt  deniity,  and  it  now  a  true 


6o6   MurrilL — The  Development  of  the  Archegoniunt 

resting  nucleus  like  that  of  the  egg.  The  membranes  of  the  two  nuclei  are  still 
intact  The  changes  in  the  two  nuclei  may  be  better  described  under  later  figures. 
In  this  case  the  stalk-cell  lies  near  the  second  sperm-nucleus. 

Fig-  39-  ( X  1600.)  The  stalk-cell  enlarged  from  the  section  represented  in 
Fig.  38  to  show  its  thick  nuclear  reticulum,  and  scanty  vacuolate  cytoplasm. 

Fig.  40.  (  X  aoo.)  This  figure  shows  the  first  sperm-nucleus  in  contact  with 
the  egg-nucleus,  and  the  second  sperm-nucleus  almost  touching  its  membrane. 
The  sperm-cells  entered  the  egg  unusually  far  down  on  its  side,  and  the  bursting  of 
the  pollen-tube  was  sufficient  to  force  them  to  this  position  near  the  egg-nucleus. 
This  also  accounts  for  the  contact  of  the  first  sperm-nucleus  at  the  side  instead 
of  the  apex.  The  remains  of  the  neck-cells  could  not  be  found.  The  egg- 
membrane  did  not  close  up  again  after  the  contents  of  the  pollen-tube  entered,  as 
was  the  case  in  the  egg  represented  in  Fig.  36. 

Fig.  41.  (X  400.)  This  represents  a  stage  succeeding  that  shown  in  Fig.  37. 
The  contents  of  the  sperm-nucleus  are  losing  their  density,  and  numerous  small 
spheres  have  appeared  in  the  nuclear  cavity.  Crenate  folds  are  observed  in  the 
membrane  of  the  egg-nucleus  at  the  surface  of  contact  of  the  two  nuclei. 

Fig.  4a.  (X  400.)  The  dense  contents  of  the  sperm-nucleus  have  disappeared 
except  at  the  centre,  and  the  nucleoli  are  larger  and  fewer  in  number.  About  the 
periphery  of  the  sperm-nucleus  a  chromatic  reticulum  is  seen.  The  chromatic 
contents  of  the  egg-nucleus  have  begun  to  migrate  to  a  point  beneath  the  sperm- 
nucleus. 

Fig.  43.  (  X  400O  The  centre  of  the  sperm-nucleus  is  quite  free  from  chromatic 
contents.  The  peripheral  reticulum  shows  larger  collections  of  chromatin. 
Nearly  all  of  the  chromatin  of  the  egg-nucleus  has  collected  beneath  the  sperm- 
nucleus.  The  crenate  folds  in  the  nuclear  membrane  of  the  egg  appear  larger, 
particularly  in  the  centre. 

F'g-  44-  C  X  400.)  The  chromatin  of  the  sperm  seems  to  have  moved  nearer 
to  that  of  the  egg.  The  intervening  membranes  are  preparing  to  break  up.  The 
egg-nncleus  has  constructed  a  chromatic  reticulum. 

Fig.  45-  ( X  400.)  The  membranes  have  broken  up,  and  the  two  nuclear 
cavities  are  continuous.  Small  spheres  with  granular  contents  now  occupy  the 
position  formerly  occupied  by  the  membranes.  The  thick  knotted  chromatic 
thread*  of  the  two  nuclei  still  remain  distinct.  All  traces  of  nucleoli  have  disap- 
peared, and  the  contents  of  the  nuclear  cavity  have  become  denser  and  more 
fibrous.  At  a  few  points,  the  outer  nuclear  membrane  is  disappearing,  and  the 
cytoplasm  is  encroaching  on  the  nuclear  cavity. 

Fig.  46.  C  X  800.)  A  tri-polar  spindle  found  near  the  apex  of  the  egg  in  the 
position  commonly  occupied  by  the  second  sperm-nucleus. 

Fig.  47.  ( X  800.)  Following  Fig.  45,  and  showing  the  origin  of  the  first 
segmentation-spindle,  which  arises  between  and  among  the  two  groups  of 
chromatin.  The  spirems  have  mostly  segmented  at  this  stage,  but  the  segmente 
have  not  yet  become  homogeneous.  The  chromatin  of  the  egg  does  not  all  appear 
in  this  section.  A  preparation  showing  the  two  groups  of  chromosomes  to.  better 
advantage  was  injured  so  that  it  could  not  be  satisfactorily  figured.  The  spindle 
is  at  first  multipolar,  but  soon  becomes  monaxial,  the  position  of  the  poles 
apparently  bemg  d«tennined  by  collections  of  a  dense  granular  substance  which 
takes  a  diffuse  reddish  sUin  with  the  Flemming  combination.    The  contents  of  the 


Viii     nil 


■*?* 


and  Fertilization  in  the  Hemlock  Spruce.    607 

nucleus  are  more  fibrous  than  before,  and  the  nuclear  membrane  has  disappeared, 
though  the  limit  of  the  nuclear  cavity  remains  the  same. 

Fig.  48.  ( X  800.)  The  spindle  is  now  monaxial  though  not  yet  distinctly 
bipolar.  *  The  chromosomes  are  homogeneous,  and  no  longer  separated  into  two 
groups.    The  contents  of  the  cavity  remain  as  shown  in  Fig.  47. 

Fig.  49.  (X  800.)  This  figure  apparently  represents  a  later  stage  than  that 
shown  in  Fig.  48,  but  no  trace  of  a  spindle  could  be  found,  even  in  very  deeply 
stained  preparations.  It  must  either  be  considered  as  a  cross-section  in  which 
the  spindle  fibres  are,  for  some  reason,  not  easily  observed,  or  the  spindle  is 
unusually  late  in  forming. 

Fig.  50.  ( X  800.)  The  spindle  of  the  first  segmentation  at  metakinesis.  The 
division  is  typical.    No  centrosomes  are  present. 

Fig.  51.     (  X  800.)    Showing  a  cross-section  of  the  same. 

f'g-  5*>  (x  »ooO  Same  as  represented  in  Fig.  50,  but  showing  a  section 
through  the  entire  egg.  The  division  is  here  oblique,  while  in  Fig.  56  it  is 
represented  as  longitudinal,  and  in  Fig.  57  as  transverse  to  the  axis  of  tlic 
archegonium. 

Fig.  S3.  (  X  800.)  The  daughter-chromosomes  of  the  first  segmentation  ap- 
proaching the  poles  in  the  form  of  U's  and  Vs.  By  counting  them  in  several 
preparations  at  this  stage,  their  number  was  found  to  be  twenty-four.  The  fibres 
of  the  central  spindle  appear  twisted  and  somewhat  thickened  at  the  equator,  but 
they  soon  disappear  without  the  formation  of  a  cell-plate. 

Fig-  64'  (><  '00.)  The  young  daughter-nuclei  resulting  from  the  first  seg- 
mentation. 

F'g-  55'    (X  'o°0    The  same  at  an  older  stage  with  the  chromatin  in  the 
'resting  condition.    The  cytoplasm  between  the  nuclei  is  finely  granular.    Note  the 
remains  of  the  ventral  canal-cell  at  the  apex  of  the  egg. 

Fig.  56.  (  X  800.)  A  spindle  of  the  second  segmentation  at  metakinesis.  It  is 
narrower  and  more  pointed  than  the  first  segmentation  spindle.  The  chromatin 
divides  in  the  same  manner,  and  there  are  no  centrosomet  present. 

F'g'  S7-  (><  '0°*)  A  fertilized  egg  containing  two  nuclei  in  simultaneous 
division.    The  stage  of  division  is  the  same  as  that  shown  in  Fig.  56. 

Fig-  58.  (  X  100.)  The  second  segmentation  is  complete,  and  the  four  resulting 
nuclei  are  equal  in  size  and  without  separating  walls. 

Fig.  59.  (  X  100.)  The  four  nuclei  are  moving  to  the  base  of  the  archegonium. 
Their  chromatic  contents  are  delicate  and  scanty.  There  is  no  special  collection 
of  fibres  about  the  nuclei.  The  archegonium  is  cut  obliquely  so  that  the  position 
and  size  of  the  nuclei  are  not  truly  represented  in  the  figure. 

Fig.  60.  (  X  100.)  The  four  nuclei  have  reached  the  base  and  are  arranged 
horizontally  in  one  plane.  A  dense  fibrous  substance  surrounds  them.  Some 
distance  above  the  plane  of  the  nuclei  is  a  zone  showing  a  regular  reticulum  quite 
ftee  from  staining  contents.  Above  this  zone  the  normal  contents  of  the  egg  are 
observed  deeply  stained. 


k&2.ti 


